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Tetrad Dissection
NEW PROTOCOL: http://openwetware.org/wiki/McClean:_Tetrad_Dissection Only follow the part that is under DIGESTION. ''' '''OLD PROTOCOL: There is a two-part training video showing S. cerevisiae tetrad dissection here and here. Also check out additional instructions for dissecting tetrads by clicking here . This link will take you to an informative pdf file online that explains general protocols for growth and manipulation of yeast. Tetrad dissection is explained under "Basic Protocol 8". In Saccharomyces cerevisiae, sporulation produces an unordered tetrad of four haploid spores which are all products of a single meiotic event, and are physically contained together in an ascus. This is immensely useful for yeast geneticists, as segregation (and linkage) of alleles can be observed directly in the spores. This is the protocol for physically dissecting yeast tetrads. It's typically preceded by sporulation, and followed by replica plating and scoring of tetrads. You'll need: Zymolyase, YPD plates, use of the 37C Shaker, and some time (up to one hour, will vary with experience) on a Dissecting Scope. 1. First, remove 200ul of your sporulated yeast in Sporulation Media to a 1.5 ml Eppendorf Tube. You can put 100ul on a slide with a cover slip to examine your yeast for tetrads before beginning digestion. Undigested tetrads will often look like three spheres stuck together in a triangle (there is a fourth behind them completing the pyramidal shape). When in doubt, go ahead and digest and look again on the plate. 1a. OPTIONAL but recommended. You can pre-dry your YPD plates at this point, to allow the yeast to dry onto plate faster after digestion. This is done in the rightmost Tissue Culture Hood only. THIS IS NOW VERBOTEN DO NOT DO THIS. If no one is using this hood, you can lift the sash until the airflow starts, place your UNSEEDED plates inside, and leave them for about 10-15 minutes to dry with their lids off. When you're finished, you'll need to remove your plates, spray down and wipe down the interior of the hood using the 95% EtOH spray bottle inside, and close the sash and be sure the UV sterlizing light comes on. Tissue culture work has priority for these hoods, so it's nice to leave a post-it with your name and cell phone # on the sash when drying plates. Never place any seeded plates, or any other source of bacteria or yeast, into these hoods. DRYING PLATES IN THE TISSUE HOOD IS NOW FORBIDDEN. 2. Using a toothpick, scoop a small amount Zymolyase from the stock. It's best to have someone show you the correct amount the first time, and when in doubt err on the side of less; you can always digest longer. Add the zymolyase to your yeast by twirling the toothpick in the 1.5ml tube. 3. Place your tube or tubes in the 37C Shaker at 200rpm for 5 minutes. NOTE BENE: OUR 37C Shaker SHOULD ALWAYS OTHERWISE BE AT 300RPM. PLEASE BE ABSOLUTELY SURE TO RETURN IT TO 300RPM AFTER YOUR DIGESTION. 4. Add 1ml of Sterile Water to your tubes to slow down further digestion and dilute the cells. Plate about 100ul of the diluted cells onto your dried YPD plates along one edge, using a coarse Sharpie to draw a line on the bottom of the plate showing where your cells are plated. 5. When the cells are dry, take your plate to the Dissecting Scope. If you've never used the dissecting scopes and micromanipulators before, you'll need a one-on-one tutorial with someone in the lab. Read additional instructions for using a dissecting scope and setting up your tetrads by clicking here . Tetrad dissection is explained under "basic protocol 8." 6. Find a tetrad- these will resemble four smaller-sized dots in a diamond pattern. If you can pick it up and put it back on the plate a couple of times without it falling apart, you can be pretty sure it's a tetrad. 7. In a grid pattern, lay out the four spores from the tetrad using the indexing knobs on the Dissecting Scope stage. You can sperate the tetrad by gently tapping the scope or stage with a finger while the tetrad is between the needle and plate. I like to lay out a grid from 0-50 in the back-fwd direction, at 35, 40, 45, and 50 in the x-7 direction, for 11 tetrads for your first couple of sets. Many people easily put twice this many on one plate in two adjacent sets of four once they get the hang of it. 8. Repeat steps 6-7 until you run out of space, tetrads, or patience. Place plates in 30C Incubator overnight.